This invention relates to the cloning, expression, sequencing, mutagenesis and functional enhancement of reactivity of recombinant monoclonal single-chain variable fragment (ScFv) antibodies against Venezuelan equine encephalitis (VEE) virus antigens.
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The alphavirus family comprises of a large number of viruses that are closely related in their molecular structure but cause a variety of different diseases in humans and other animals [1]. Some alphaviruses, upon infection, enter the central nervous system (CNS) and lead to encephalitis. A New World Alpha virus of particular importance in this regard is Venezuelan equine encephalitis (VEE) virus. VEE virus infections mainly target the CNS and lymphoid tissues causing severe encephalitis in equines and systemic febrile infection with occasional encephalitis in humans. VEE virus is highly infectious by aerosol inhalation for humans [1].
Serologically the VEE complex of viruses can be subdivided into six subtypes (I-VI), with subtype I exhibiting five variants (IAB, IC, ID, IE, IF) [1]. VEE epizootics are associated with members of subtypes IAB or IC. The other subtype I variants (ID, IE and IF) and subtypes II-VI have been associated with enzootic VEE transmission [2].
The molecular structure of the VEE virion consists of a plus sense RNA genome encapsulated in an enveloped icosahedral nucleocapsid [3]. The envelope contains two important structural glycoproteins (gp), E2 (56 KDa) and E1 (50 KDa) [4]. The viral neutralization sites reside in the E2 envelope protein [5]. Thus the E2 protein of VEE is an important target for immunodetection/protection studies.
Hybridoma technology [6] made it possible to generate monoclonal antibodies (Mab) directed against viruses. The disadvantages of using monoclonal antibodies (Mabs) as immunodiagnostic or immunotherapeutic reagents are known. The cost of large-scale production of Mabs is excessive. The potential for genetic variations introduced during repeated cycles of cell growth make Mabs difficult to handle and potentially unreliable. In addition, antigenicity of the complete antibody molecule, when administered as therapeutic reagent, is associated with xe2x80x9cserum sicknessxe2x80x9d in recipients. Furthermore, due to the large size of the whole antibody molecule, there is low penetrability of administered antibody into target tissues. These features make the complete antibody molecule unattractive for use as therapeutic reagent [19, 20].
However, with the development of recombinant antibody technology, where functional antibody fragments can be produced in bacteria, the application of antibodies as immunodiagnostic and/or immunotherapeutic reagents has become more feasible [21, 22, 23, 24].
To develop a monoclonal recombinant antibody from 1A4A-1 Mab, the inventors chose the Recombinant Phage Antibody System (RPAS). Briefly, in RPAS technology, the Variable heavy (VH) and Variable light chain (VL) domains of an antibody are cloned and covalently joined by a polypeptide linker in bacterial expression vectors generating single chain Variable fragment (ScFv) antibodies [8, 9, 10]. This results in expression in bacteria of the antigenic recognition domain of an antibody as a single chain, which can fold itself into a functional molecule. The attractiveness of this system is in the relative ease with which huge quantities of functionally active molecules can be obtained in a very short time. Since these ScFv lack the constant region of the antibody molecule, they have very low antigenicity. The low antigenicity of ScFv, coupled with their small size (xcx9c30 KDa) and hence the ease with which they can penetrate tissues, makes these molecules attractive alternatives to whole antibody molecules as therapeutic materials.
Roehrig et al. [7] generated Mab directed against VEE viruses. Using these Mab, the antigenic structure of the envelope glycoprotein E2 of VEE virus was analyzed. Protection studies using anti-E2 VEE virus Mab revealed a critical neutralization epitope (E2c). One of the Mab that recognized this neutralization epitope (E2c) was 1A4A-1. Mab 1A4A-1 not only recognizes VEE virus serological subtypes IA-ID and IIEVE in enzyme-linked immunosorbant assay (ELISA) but also can neutralize all of these subtypes [7], making it a very good candidate for development of second generation antibody with both diagnostic and immunotherapeutic application potential.
The present invention is directed to the cloning, expression, sequencing, mutagensis, and functional enhancement and of ScFv from hybridoma cell line 1A4A1.
The initial results of generating a recombinant 1A4A-1 ScFv antibody are described elsewhere [11]. Briefly, 1A4A-1 Mab was cloned as a ScFv antibody at Defence Research Establishment Suffield (DRES). The functional analysis of the 1A4A-1 ScFv (A116) by ELISA and western blotting revealed low reactivity to VEE whole virus antigen. Sequence analysis of A116 revealed three base deletions in the 5 prime end of the A116 VL gene. At the protein level, these mutations resided in the framework-1 region of VL of A116. The three individual base deletions introduced a localized frame shift in the first 20 amino acids of the conserved framework-1 region of VL of A116, resulting in a partially functional protein [11].
Framework-1 region of the light chain is highly conserved in antibody molecules. It adopts an anti-parallel beta pleated sheet conformation. xcex2-strands A and B of the antibody light chain stabilize each other through hydrogen bonding and ultimately place complementarity determining region (CDR) L1 in proper context in relation to the other two CDRs for antigen interaction. Since the frame shift in VL of A116 occurred in a region that is conserved in antibody molecules, it is possible that the frame-shift affected the overall conformation of the antibody molecule, thus rendering it partially inactive. A repair strategy was conceived whereby the framework-1 region would be restored to consensus amino acid sequence, and such repaired clones could be analysed for restored reactivity to VEE whole virus antigen. In this invention, the inventors describe the successful isolation of MA116 clones with enhanced reactivity to VEE whole virus antigen and discuss the molecular changes that restored the full functionality of original A116 ScFv protein.
Through repairing the genetic mutation of the ScFv antibody gene, the present invention provides enhanced reactivity of the cloned ScFv antibody with homologous VEE antigen.